Project Summary The elderly population is expected to double in the next 40 years, and therefore aging-related diseases are likely to become a major healthcare burden. Lung diseases, including emphysema, chronic obstructive pulmonary disease, and interstitial lung disease, and general lung pathologies including acute lung injury (ALI) and the associated acute respiratory distress syndrome (ARDS), increase dramatically in the elderly, although the underlying causes and mechanisms remain undefined. Importantly, aging is associated with a number of changes in lung physiology and lung immunity, including a decreased vital capacity, decreased mucociliary clearance, and increased susceptibility to both bacterial and viral infections. Changes in lung physiology and pathogen susceptibility may be related; however, there is little known regarding the impact of these age-related physiologic changes on surveillance of the lung by the immune system. Identifying molecular changes in these processes with age can lead to new therapeutic interventions to help protect the aging lung and reduce disease incidence. We have established a novel human tissue resource where we obtain multiple lymphoid and mucosal tissues from organ donor of all ages, including all ages of adulthood up to the 9th decade of life. Importantly, we obtain lungs, lung-associated and peripheral lymphoid tissues, enabling novel study of aging- associated changes over a continuum of decades. Our central hypothesis is that with age, there is decreased immune surveillance and protection both from lung-resident immune cells and lung-associated lymph nodes, resulting in decreased lung cell integrity and increased susceptibility to damage. In the proposed study, we will take a multi-disciplinary approach to study the interaction between the lung immune system and lung epithelium as a function of age. In aim 1 we will identify how lung resident immune cells and lung-associated lymph nodes (LN) alter with age. We will use high dimensional cellular and molecular profiling using high parameter flow cytometry and whole transcriptome profiling to assess changes in immune cell populations, and immunofluorescence imaging to determine the frequency and localization of lung-resident immune cells, and the functional activity of LN through examination of LN follicles. In aim 2, we will use real time optical imaging of the human lung to determine the effect of age on the quality of the alveolar-capillary fluid barrier, mitochondrial function in the alveolar epithelium, and the extent to which resident AMs maintain gap junctional and paracrine communication with the alveolar epithelium. In aim 3, we will define the age-drive gene expression program in all lung cells, using single cell RNA-Seq to assess how gene expression programs that define lung epithelial cells, endothelial cells, innate and adaptive immune cells will be altered and the kinetics of these alterations. The proposed studies will reveal new insights into human lung immunology and physiology and mechanisms for age-associated changes that predispose individuals to increased respiratory disease.